1. Field of the Invention
This invention relates to a device and method for culturing pathogenic microorganisms and viruses to conserve laboratory space and avoid contamination of the culture and release of pathogens into the environment. In particular, this device pertains to a device and method for obtaining large volumes of pathogenic viruses such as the AIDS virus in a closed culture system.
2. Description of the Related Art
Pathogenic animal viruses, such as the human immunodeficiency virus (HIV), the rabies and herpes viruses, and pathogenic bacteria such as Neisseria meningiditis and Mycobacteria avium must be studied with extreme precaution to avoid spread of the virus and contamination of workers and research areas. In the following discussion of viruses, it is understood that other pathogens may be handled in analogous fashion. The problem is that in order to study these viruses, large quantities of viruses and large volumes of virus extracts must be prepared and isolated from growth media and contaminating cells, microbes and debris. Although other pathogens, such as pathogenic bacteria or yeasts do not usually require such large volumes of cell growth as are required for viruses to obtain sufficient material for study, the cells must also be cultured in quantity and handled with great care to avoid worker exposure and accidental release of the organisms.
Microbial cellular pathogens of animals such as cellular viruses generally must be cultured in growing animal cell cultures. The standard method to culture such cells is by the use of roller bottles. Roller bottles are coated on the inside by a layer of solid or semi-solid growth substrate bathed by a nutrient medium on which the cells are grown. The cell growth in roller bottles and similar growth vessels, and thus, the viral yield, are limited by the the internal surface area of the glass or plastic bottle and by the nutrients in the nutrient medium. To obtain the large scale viral yields that are necessary to study viruses, the number of roller bottles needed may often fill shelves that extend from wall to wall and floor to ceiling in several growth rooms. This requires that large areas of laboratory space be allocated for incubation facilities and often necessitates construction and/or room conversion and attendant delays for those beginning to do research in this area.
The processes of preparing the large number of roller bottles, inoculating them with infected cells, incubating them under the appropriate conditions, extracting the viruses from the individual bottles, concentrating the viral suspensions, and lysing the viruses to obtain non-pathogenic viral extracts for research also are time-consuming and expensive. The numerous complicated manual manipulations required in these procedures allow many opportunities for mistakes to occur and problems to develop. Errors by the laboratory workers may result in unwarranted experimental conclusions and/or increased expenses and delays occasioned by the repeated experiments.
The multiple experimental steps involving opening of the roller bottles and the multiple number of roller bottles required increases the likelihood that some of the human host cells will be contaminated by undesirable microorganisms or viruses that may kill the host cells. This may decrease viral production of the system being studied or complicate extraction of the desired virus components from the culture.
The large volumes of viral suspensions produced in the existing culture systems necessitates a massive time-consuming effort of viral harvesting. The dilute viral suspensions that are obtained from the prior culture systems are difficult to treat effectively to lyse the viruses until the suspension has been concentrated. Thus, in a typical procedure, these large volumes of infective viruses must be placed in centrifuge tubes. The supernatant fluid must be decanted, with the possibility of resuspension of the infective particles, and the pellet must then be treated to lyse the viruses.
Thus, the numerous manual procedures that are required by the prior art increases the possibility of escape of some of the pathogenic virus particles from the culture system and their release from the laboratory into the environment. It is of the highest priority that the release of pathogenic viruses such as the AIDS virus be avoided.
Mass transfer operations are often used to attempt to solve some of the problems of growing a large number of cells that are associated with culture systems such as roller bottles. In the mass transfer chamber, a biological medium may be on one side of a mass transfer surface element and the medium to which or from which mass transfer is to be effected is disposed on the opposite side of the element. Counterflow of the two media past each other effects diffusional and/or osmotic mass transfer. Problems in the prior art, which include reduction of mass transfer after prolonged operation of a mass transfer element, longitudinal decrease of mass transfer efficiency due to fouling of mass transfer surfaces, and presence of undesirable micro-environments and areas of preferential cell growth, are substantially avoided with the cell culturing system disclosed in my copending patent application U.S. Ser. No. 06/936,486 filed Nov. 26, 1986, the entire disclosure of which is hereby incorporated by reference. This system may be used to culture cells continuously on cellular or microbead substrates with a minimum of risk.
In the culture of host cells to produce progeny viruses, even with optimized mass cell culturing systems, there remain problems of handling the culture fluid containing the viruses that is being circulated through the system. Inhibitory substances of various sizes must be removed from the media without removing the viruses to allow cell growth to continue and viral concentration to increase. There also must be a way to add new medium in order to allow cell growth to continue without increasing the volume of the culture medium that contains the virus. These purposes may be accomplished by use of the stacked filter train disclosed in my copending patent application U.S. Ser. No. 07/104,177, filed Oct. 2, 1987, the entire disclosure of which is hereby incorporated by reference, which allows media to be added or wastes to be withdrawn from a cell culture system without contamination of the system or the operator.
Even with the prior art technology, the problems remain of finding a method of culture of the cells to maximize virus yield; an arrangement of culture vessel apparatus and stacked filter train(s) to allow optimal recovery of viruses without excessive contamination by metabolites, medium components, cell debris, or other unwanted materials; and most importantly, a means by which viruses may be concentrated and lysed without causing them to be removed from the culture system to avoid all handling of the pathogenic viruses after the initial inoculation of the system.
Accordingly, it is an object of the present invention to provide an improved method and apparatus for effecting increased cell growth and pathogen yield in which release of infective pathogens is avoided.
It is another object of the invention to provided an improved method and apparatus for effecting increased cell growth and pathogen yield in which the infective pathogentacre not handled by laboratory personnel after the original inoculation with the infected cells.
It is another object of the invention to provide an improved method and apparatus for effecting increased cell growth and pathogen yield by aseptic removal of spent medium components.
It is another object of the invention to provide an improved method and apparatus for effecting increased growth and pathogen yield by aseptic addition of nutrients.
It is a further object of the invention to provide a method and apparatus for effecting increased pathogen yield that occupies a minimum of laboratory space and does not require construction of special rooms or buildings.
It is another object of the invention to provide a method and apparatus for concentrating pathogen suspensions such as viral suspensions for subsequent lysis without requiring handling large volumes of infective pathogens.
It is another object of the invention to provide a cost-reducing method and apparatus for effecting increased cell growth and pathogen yield.
It is a further object of the invention to provide a sealed virus-tight, unified, preassembled apparatus for aseptically growing pathogens, monitoring and adjusting medium components, harvesting the pathogens from the medium, rendering the pathogens harmless and obtaining unhazardous pathogens or pathogen components.
Other objects and advantages of the invention will be more fully apparent from the following disclosure and appended claims.